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IDH gene dsRNA capable of improving termite control effect of metarhizium anisopliae

A technology for Metarhizium anisopliae and termites, which is applied in the directions of DNA/RNA fragments, DNA preparation, recombinant DNA technology, etc., can solve the limitation of the promotion and application of termites, the unsatisfactory effect of Metarhizium anisopliae on termite field control, and Metarhizium anisopliae. Reduced lethal effects, etc.

Active Publication Date: 2020-10-30
宜昌市金通白蚁防治有限公司 +1
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese termite control workers have done a lot of work on the use of Metarhizium anisopliae to control termites. However, because termites are social insects that live in groups, individuals can perform hygienic behaviors such as grooming, feeding, isolation, and burial. Pathogen infection can activate physiological systems such as autoimmunity, detoxification, and anti-oxidation. These behaviors and physiological immune strategies have led to unsatisfactory field control effects of Metarhizium anisopliae on termites, which severely limits the promotion and application of termite biological control technology.
[0004] The inventor’s previous work found that Metarhizium anisopliae has a strong lethal effect on single termites, but with the increase of termite density, the lethal effect of Metarhizium anisopliae significantly decreases

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] ds IDH The preparation process:

[0027] (1) Total RNA was extracted from the body of Termites termites and Termites cerevisiae, and the termite cDNA library was obtained by reverse transcription.

[0028] (2) Using termite cDNA as a template, using IDH Specific primers (P. black-breasted termites: upstream primer 5'-AGG CTGCGA TTG AGT CTG-3' and downstream primer 5'-ACC AGC ACA CAT ATC AGA C-3'; black-winged soil termites: upstream primer 5'-ACC CTG TCG ATC ACT GGA AG-3' and downstream primer 5'-TTG CTT TAT CTT GGC CTG CT-3'), amplified by PCR IDH Gene fragments, followed by separation and purification by agarose gel electrophoresis IDH Gene fragment.

[0029] (3) By T-A cloning the IDH The gene fragment was introduced into the pMD 18-T plasmid (Baoric Medical Biotechnology (Beijing) Co., Ltd. (takara China)), and the target gene was obtained by shaking bacteria detection IDH of pure plasmids.

[0030] (4) With the above IDH The plasmid is used as a template, u...

Embodiment 2

[0037] ds IDH The implementation process 1:

[0038] 1.5 μg corresponding to ds by microinjection IDH Introduced into the body of Termites termites, cultured in a 3.5 cm diameter petri dish covered with wet filter paper for 2 days, as ds IDH Treatment group; 1.5 μg ds by microinjection GFP Introduced into the body of Termites termites, cultured in a 3.5 cm diameter petri dish covered with wet filter paper for 2 days, as ds GFP control group.

[0039] After 2 days, using Metarhizium anisopliae (concentration 10 9 spores / mL) to infect one termite, and at the same time put 5 termites that had been injected with the above dsRNA (ds IDH or ds GFP ) of termites. Therefore, the experiment included 2 treatment groups: (1) 1 infected termite + 5 ds-injected IDH Termites; (2) 1 infected termite + 5 injected with ds GFP termite. After 1 day of co-cultivation, the injected termites in the group were all infected with a small amount of spores of Metarhizium anisopliae due to ...

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Abstract

The invention relates to IDH gene dsRNA capable of improving the termite control effect of metarhizium anisopliae. The sequence design of the dsRNA is derived from a key metabolic enzyme gene, namelyan isocitrate dehydrogenase (IDH) gene, in bodies of the eupolyphaga nigromaculata and the termites nigromaculata. An IDH dsRNA template is synthesized by utilizing a specific primer of the IDH gene,and then dsIDH is generated through transcription of a T7 in-vitro transcription system. After dsIDH is introduced into a termite body by adopting an RNAi technology, the IDH gene expression is reduced, the antifungal activity of the termite is remarkably reduced, the apoptosis rate is increased, the infection level is increased, the IDH activity is reduced, the ATP level is reduced, the movementdistance is reduced, and the death rate of the termite is remarkably increased. The dsIDH can significantly improve the control effect of metarhizium anisopliae on termites, is environmentally friendly and safe to people and livestock, and has good research and application prospects.

Description

technical field [0001] The invention relates to a method capable of improving the termite control effect of Metarhizium anisopliae. IDH Gene dsRNA. Background technique [0002] Termites are an ancient social harmful insect that can harm agricultural and forestry crops, garden trees, housing construction and water conservancy projects in my country. They are known as "toothless tigers" and seriously threaten people's lives and property safety. In my country, except for a few provinces (regions) such as Xinjiang, Qinghai, Ningxia, Inner Mongolia and Heilongjiang, termites are distributed in other provinces (regions). The economic loss caused by termite damage in our country reaches 10 billion yuan every year, so termite control work is highly valued. At present, the methods of controlling termites include physical control, chemical control, biological control and other methods, among which chemical control is the main means of termite control. Currently, many highly toxic ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10A01N57/16A01N63/30A01P7/04
CPCA01N57/16A01N63/30C12N15/1137
Inventor 黄求应刘龙徐焕雷朝亮赵星颖
Owner 宜昌市金通白蚁防治有限公司
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